Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis.
The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-l-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.
Woodcock
S C
SC
Department of Molecular Genetics, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, U.K.
Raux
E
E
Levillayer
F
F
Thermes
C
C
Rambach
A
A
Warren
M J
MJ
D000595:Amino Acid Sequence; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D006418:Heme / Q000031:analogs & derivatives / Q000096:biosynthesis; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D012436:S-Adenosylmethionine / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000096:biosynthesis
C012851:cobyric acid; D006418:Heme; C009272:siroheme; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase; D014805:Vitamin B 12